POSTTRANSLATIONAL AND GENETIC ORIGINS OF TOXIN DIVERSITY

毒素多样性的翻译后和遗传起源

• 批准号: 6564575
• 负责人: DAVID R HILLYARD
• 金额: $ 14.53万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564575
• 关键词: Gastropoda; affinity chromatography; animal poison; biochemical evolution; calcium channel blockers; carboxylation; conotoxin; gene expression; genetic library; molecular cloning; molecular genetics; neuropeptides; neurotoxins; nucleic acid sequence; nucleic acid structure; posttranslational modifications; protein purification

Conus snails synthesize a bewildering variety of oligopeptides which target neuronal receptors and ion channels. The peptides contain an invariant framework of disulfides, while the remaining amino acid residues are relatively random and contain a gamut of post-translational modifications which are essential for their biological activity. Conus have evolved an efficient genetic means for encoding the variations. One of our goals is to understand how this diversity is generated. This will be achieved by determining the structure and sequence of the known conotoxin genes and their cDNAs. We will also attempt to characterize additional conotoxins by sequencing of selected cDNAs obtained from venom duct after elimination by subtractive hybridization of those that have been previously identified. We hope this knowledge will enable use to design efficient combinatorial methods for obtaining novel ligands of pharmacological import. A second goal of the project is to understand the mechanism of post- translational modification of the conotoxins, especially how gamma- carboxylation of glutamate is achieved. This modification is found in some conotoxins and in all conantokins, and is essential to their activity. Conus are the only non-mammalian species known to elaborate this modification. In mammals, gamma-carboxylated proteins are vital for the homeostasis of the blood coagulation cascade. Our aim is identify and characterize the enzymes necessary for gamma-carboxylation. This will be done by biochemical isolation of the enzyme and cloning of the gene. We hope the Conus gamma-carboxylation system is sufficiently similar to the mammalian system, and will also afford insight for the synthesis of active coagulation factors.

圆锥蜗牛合成了令人困惑的各种寡肽 靶向神经元受体和离子通道。肽包含一个 二硫化物的不变框架，而其余的氨基酸残基 相对随机，包含一系列翻译 对其生物活性至关重要的修改。圆锥 已经进化了一种编码变化的有效遗传手段。一 我们的目标是了解如何产生这种多样性。这会 可以通过确定已知的结构和序列来实现 结合毒素基因及其cDNA。我们还将尝试表征 通过测序从毒液中获得的选定cDNA进行的其他结合毒素 消除后通过减法杂交的导管 以前已确定。我们希望这些知识能够使用 设计有效的组合方法，用于获得新的配体 药理进口。 该项目的第二个目标是了解后的机理 结合毒素的翻译修饰，尤其是γ- 实现了谷氨酸的羧化。在某些人中发现了这种修改 结合毒素和所有Conantokins，对于它们的活性至关重要。 圆锥是唯一已知的非哺乳动物物种 修改。在哺乳动物中，γ-羧化蛋白对于 血液凝结级联的稳态。我们的目标是确定和 表征伽马羧化所需的酶。这将是 通过酶的生化分离和基因克隆进行。我们 希望Conusγ-羧化系统与 哺乳动物系统，也将为主动的综合提供见解 凝血因子。